B cell function is dependent upon the ability of the membrane B cell antigen receptor (BCR) to bind antigen and to effectively induce an efficient cascade of biochemical events from the membrane to the nucleus. These events culminate in the cytosol to rearrange the morphology of the cell through cytoskeletal reorganization and in the nucleus to activate the transcription of new genes to promote cellular differentiation and proliferation. These biochemical and cellular events are required for the ability of B cells to mature and function to produce an efficient immune response to foreign pathogens. Conversely, abberant activation of B cells can lead to unregulated cellular proliferation and uncontrolled clonal expansion, resulting in B cell tumors. In addition, unregulated activation of B cells may also contribute to a variety of autoimmune diseases mediated by self-reactive antibodies. Thus, the ability to modulate BCR mediated signaling events may provide a rational approach in the treatment of B-cell mediated tumors and also in autoimmune diseases in which abberant B cell activation may occur.
Ligand binding of the BCR to antigen activates at least two major distinct biochemical pathways within the cell. The first results in increases in intracellular calcium levels that is mediated by an enzyme known as .gamma. isoform of phospholipase C (PLC-.gamma.). Two forms of PLC-.gamma., .gamma.-1 and .gamma.-2, appear to be capable of mediating this calcium response. The second major biochemical pathway activated through antigen engagement of the BCR is the activation of the ras pathway. Activation of this pathway appears to be mediated by a molecule known as Grb2. Grb2 is an adapter molecule containing two SH3 domains that mediate its interaction with the guanine nucleotide exchange factor, Son of Sevenless (SoS) which in turn activates the ras pathway by facilitating the exchange of GDP for GTP on the ras molecule. Activation of both the ras and calcium pathways are required for efficient BCR function. The molecular mechanisms by which Grb2 and PLC-.gamma. become activated by the BCR remain unclear at this time.
A third, though less characterized pathway, activated by the BCR is cytoskeletal rearrangement and aggregation of the BCR (a phenomenon known as capping). The molecular mechanisms of this pathway are unclear.
While the biochemical mechanisms by which the calcium and ras signaling pathways become activated remain unclear, studies have demonstrated that both of these pathways require the activation at least two families of protein tyrosine kinases (PTKs). The first family is the Src-family of PTKs which can associate with the BCR. A second family is the Syk PTK which interacts with activated, phosphorylated BCR. Abrogation of the function of either Src-PTKs or Syk interferes with calcium and ras signaling pathways and results in a non-functional BCR.
Thus, the discovery of molecules which interact with either Grb2 or PLC-.gamma., and thus play a role in the regulation of the ras and calcium signaling pathways are desired. Accordingly, it is an object of the present invention to provide such molecules, termed "BLNK" proteins, and to provide methods of using such molecules in screening assays.